Molecular display technologies are widely used to screen for potential affinity binders to a specific target molecule, however, there is potential for improving thereon. For example, phage display antibody technologies are used for isolating antibody fragments specific to antigens of interest, but selection of libraries against cell-surface antigens remains very challenging. The heterogeneity of the cell-surface and, accordingly, the relatively low concentration of the target antigen, give rise to large numbers of background phage clones. These phage clones may be non-specific binding clones, or may be specific for antigens other than the desired cell-surface target. Consequently, poor enrichment for binding phage clones is typically observed in cell selections. However, many proteins require the membrane environment for proper folding and stability and, as such, the ability to select phage-displayed antibody libraries against cell-surface epitopes remains crucial. If a protein is not properly folded, certain epitopes may not be available for binding by, for example, an affinity reagent. Likewise, proteins that are part of large complexes or associated with DNA, histones or other subcellular structures contain epitopes that are not necessarily made available for binding following traditional purification methods. For example, the properties of multi-pass membrane G-protein coupled receptors make their expression and purification very difficult, yet they are particularly relevant drug targets [1, 2]. Indeed, the high specificity of monoclonal antibodies, combined with their ability to engage immune mechanisms, makes this class of biologics of particular interest in the treatment of numerous cancers and infectious diseases [3,4,5]. A reliable selection methodology for targeting exposed epitopes (e.g. cell-surface epitopes), which eliminates the need for highly purified antigens, would significantly expand the range of antigens that could be targeted by therapeutic monoclonal antibodies.
Phage display selection strategies to reduce background binding to cells have included negative or competitive pre-absorption steps against multiple cell-lines [6, 7, 8, 9, 10] and various strategies to remove unbound from bound phage, including centrifugation through a density gradient [11, 12] and the pathfinder approach [13, 14]. Although these methods may help to enrich for phage clones specific to the antigen of interest, the number of unique antibody fragments recovered by these methods often remains relatively low, as phage display methodologies typically exhibit an affinity based selection pressure that promotes sequence convergence in later rounds of selection. New strategies are required to identify less prevalent clones that may exhibit desirable binding properties.